Reticulon-like-1, the Drosophila orthologue of the hereditary spastic paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons

Abstract

Several causative genes for hereditary spastic paraplegia encode proteins with intramembrane hairpin loops that contribute to the curvature of the endoplasmic reticulum (ER), but the relevance of this function to axonal degeneration is not understood. One of these genes is reticulon2. In contrast to mammals, Drosophila has only one widely expressed reticulon orthologue, Rtnl1, and we therefore used Drosophila to test its importance for ER organization and axonal function. Rtnl1 distribution overlapped with that of the ER, but in contrast to the rough ER, was enriched in axons. The loss of Rtnl1 led to the expansion of the rough or sheet ER in larval epidermis and elevated levels of ER stress. It also caused abnormalities specifically within distal portions of longer motor axons and in their presynaptic terminals, including disruption of the smooth ER (SER), the microtubule cytoskeleton and mitochondria. In contrast, proximal axon portions appeared unaffected. Our results provide direct evidence for reticulon function in the organization of the SER in distal longer axons, and support a model in which spastic paraplegia can be caused by impairment of axonal the SER. Our data provide a route to further understanding of both the role of the SER in axons and the pathological consequences of the impairment of this compartment.

Figure 1.

Loss of Drosophila reticulon causes a progressive locomotor deficit. (A) Dendrogram showing similarity of human and Drosophila reticulon proteins, drawn from a Clustal alignment using the neighbour-joining algorithm in MEGA 5.05. Dashed line indicates fast divergence of Drosophila Rtnl2. (B) The Rtnl1 locus showing proteins encoded by transcripts A-R (Flybase Genome Browser R5.41), location of VDRC RNAi fragments (RNAi) and the Rtnl1::YFP insertion (CPTI00291). (C–E) Confocal sections showing localization of Rtnl1::YFP in epidermal cells overlapping with KDEL (arrowheads in inset; C), in the larval ventral nerve cord (D) where it localizes preferentially in neuropil (arrow) and nerve bundles (arrowheads) in contrast to KDEL, which is concentrated within cell bodies (boxes), and in NMJ presynaptic boutons (E), where it follows the path of the axonal MT marker Futsch (upper panel). (F) VDRC RNAi line (construct GD900) successfully knocks down Rtnl1 expression as observed by PCR amplification of Rp49 and Rtnl1 cDNA from progeny of da-GAL4 crossed to either w¹¹¹⁸ (control) or Rtnl1 RNAi flies. (G and H) Loss of Rtnl1 causes an age-dependent climbing deficit. Graphs represent end-to-end distance travelled in 15 s (G) and climbing velocity (H) of Rtnl1 RNAi and control flies (mean ± SEM here and in all subsequent graphs; *P < 0.05, **P < 0.01, ***P < 0.005, otherwise P > 0.05; two-way ANOVA and Bonferroni post-tests; n = 3 independent experiments).

Figure 2.

Loss of Rtnl1 causes expansion of ER sheets and increases the ER stress response. (A) KDEL distribution in third-instar larval epidermal cells is reticular in control larvae but more diffuse in Rtnl1 RNAi larvae; distribution of the Golgi marker GM130 is broadly unaffected. Graph shows KDEL staining intensity per cell as a percentage of control levels (n= 4 independent experiments; ns, not significant, P > 0.7). (B) Electron micrographs show increased length of ER sheets in Rtnl1 RNAi epidermal cells compared with controls. Arrowheads, ER sheets; Nuc, nucleus; M, mitochondrion. The graph shows the quantification of ER sheet cross-sectional length (***P < 0.005; n = 3 independent larvae). (C and D) Single-confocal sections of larval epidermal cells (C) and ventral nerve cord (D) show increased ER stress, measured by increased Xbp1::GFP expression in Rtnl1 RNAi larvae compared with controls and quantified in graphs (**P < 0.01, ***P < 0.005; n = 12–16).

Figure 3.

Rtnl1 interacts with the Acyl-CoA synthetase Acsl. Confocal sections showing overlapping localization of Rtnl1::YFP and Acsl in axons (linear staining indicated with arrowheads; A) and epidermal cells (B). (C) Protein lysates from OregonR (control) and Rtnl1::YFP flies were immunoprecipitated with Strep-Tactin Sepharose. Whole lysate (In), unbound (UB) and bound (Out) fractions were analysed by western blot. Loss of Rtnl1 reduces endogenous Acsl protein levels relative to controls in immunoblots of adult lysates (D) and single-confocal sections of larval epidermal cells (E). (F) Anti-Acsl staining of OK6GAL4;UAS-Acsl::myc motor neurons labels structures running along axons (arrowheads). Acsl staining is significantly lowered in posterior but not anterior segments of long motor axons upon knockdown of Rtnl1 (F). Graphs represent Acsl expression levels (ns, not significant, P > 0.2; **P < 0.01; n = 6–8 lysates in D, n = 14–16 larvae in E and F).

Figure 4.

Rtnl1 interacts with the MT-associated protein Futsch. (A) Protein lysates from OregonR (control) and Rtnl1::YFP flies were immunoprecipitated and blotted as in Figure 3A. Loss of Rtnl1 reduces Futsch protein levels relative to controls in immunoblots of adult lysates (B) and in posterior but not anterior segments of long motor axons (C). Graphs represent Futsch expression levels (ns, not significant, P > 0.44; *P < 0.05, ***P < 0.001; n = 6–8 lysates in B, n = 16–19 larvae in C).

Figure 5.

Effects of Rtnl1 loss on motor neuron mitochondria. (A) Single-confocal sections showing no effect of Rtnl1 RNAi (generated using OK6-GAL4 in this figure) on mito::GFP and CSP in posterior larval motor axons. (B and C) Single-confocal sections showing mito::GFP (green) and Dlg (magenta) at 1b boutons of anterior (B) and posterior (C) abdominal NMJs from control and Rtnl1 RNAi larvae. Graphs show the number (in 1b and 1s boutons) and the size (in 1b boutons) of mitochondria in posterior NMJs (ns, not significant; P > 0.5, **P < 0.01, ***P < 0.0005; n = 18–22 NMJs for 1b boutons, n = 12 NMJs for 1s boutons).

Figure 6.

Model of hairpin loop protein dysfunction in motor neuron axonopathy. (A) The schematic diagram represents motor neurons under ‘normal’ or non-diseased conditions. Ribosomal studded RER sheets predominate within the neuronal cell body, whereas tubular SER runs the length of the axon closely associated with the MT cytoskeleton (13,21,29). In addition, the ER has extensive contact points with mitochondria which are required for lipid biosynthesis, calcium homeostasis and mitochondrial division (38,46). (B) Mutation or depletion of any one of the hairpin loop proteins causes disruption of the ER network (either less extensive and/or discontinuous) and degeneration of long motor axons as observed in HSP. Our findings have revealed an increased susceptibility of posterior axons to the disruption of ER organization, resulting in the disruption of tubular SER, MT cytoskeleton and mitochondria. This suggests that the loss of tubular ER from long motor axons may be the mechanism by which the loss of hairpin loop protein function gives rise to motor neuron axonopathy.